1. Technical Field
The subject invention relates generally to immunoassays for detection of antibodies by use of chemiluminescent compounds. More particularly, the subject invention relates to chemiluminescent immunoassays to detect antibodies wherein a precomplex mixture is created and a two-step assay is performed resulting in a greater or comparable signal as compared to a three-step assay.
2. Background Information
Immunoassays that employ chemiluminescent labels as the signal generating compound are known. For example, the application of chemiluminescence generation and detection for immunoassays has been reviewed by W. R. Seitz, "Immunoassay Labels Based on Chemiluminescence and Bioluminescence," Clinical Biochemistry 17:120-126 (1984).
A method for performing a chemiluminescent assay involving directly exciting and measuring a chemiluminescent signal emanating off an immune complex immobilized on or in a solid, porous element that is used as a separation means in a heterologous immunoassay and an apparatus for performing this measurement are described in U.S. Pat. No. 5,089,424 and now abandoned U.S. patent application Ser. No. 07/206,645 which both enjoy common ownership and are incorporated herein by referenece.
Additionally, a method for determining the presence of an analyte, in particular, HCV antibody, in a test sample by specific amplification of a chemiluminescent signal generated from a heterogeneous immunoassay which utilizes a precomplex is described in U.S. Pat. No. 5,705,330 which is herein incorporated in its entirety by reference.
The generation of light as a result of a chemical reaction is known in the art and was reviewed by Schuster and Schmidt in "Chemiluminescence of Organic Compounds," V. Gold and D. Bethel, eds., Advances in Physical Organic Chemistry 18:187-238, Academic Press, New York (1982). The use of acridinium compounds as labels for immunoassays and subsequent generation of short-lived chemiluminescence signals from these labels has been described in Weeks et al., in "Acridinium Esters as Highly Specific Activity Labels in Immunoassays," Clin. Chemistry 19:1474-1478 (1984). The use of stable acridinium sulfonamide esters has been described in a co-owned patent by Mattingly et al., U.S. Pat. No. 5,224,833 incorporated herein by reference and published as European Patent Appln. No. 0273115. The generation of long-lived luminescent signals has been described in the art as resulting from action of enzymes or nucleophilic agents on dioxetane compounds containing an adamantane structure. See, for example, European Patent Appln. No. 0254051; published PCT Patent Appln. No. WO 8906650; Bronstein et al., J. Bioluminescence and Chemiluminescence 4:99 (1988) and 5.sup.th International Conference on Biolumin. and Chemilumin., Florence-Bologna, Italy, Sep., 25-29 (1988). The use of a signal enhancer such as the use of avidin-biotin is also known. For example, U.S. Pat. No. 4,228,237 describes the use of a biotin-labeled specific binding substance for a ligand used in a method which also employs an enzyme labeled with avidin. The use of a biotin-avidin-biotin system is described in U.S. patent appln. Ser. No. 608,849 filed on May 10, 1984, now abandoned, which enjoys common ownership and is incorporated herein by reference (published on Nov. 13, 1985 as European Patent Appln. Ser. No. 160900). Methods of enhancing and amplifying the chemiluminescent signal generated in an immunoassay are known in the art. For example, U.S. Pat. No. 4,927,769 describes a method of enhancing the chemiluminescent signal generated from acridinium-ester labeled conjugates by the addition of surfactants. Also, U.S. Pat. No. 4,959,182 describes a method for amplifying the chemiluminescent signal generated from alkaline phosphatase-catalyzed 1,2-dioxetanes by the addition of a surfactant and a fluorescent compound attached to it.
Known traditional methods for performing chemiluminescence assays for detection of antibodies, if utilizing enhanced compounds as herein described, usually involve separated incubation steps for reacting the sample and capture reagent, reacting the sample/capture mixture with the conjugate to which it is attached and enhancer compound, and reacting the sample/capture/conjugate mixture with an enhancer-specific binding member, and then generating a signal.
In the present invention, it has been discovered that, by forming a precomplex of conjugate and probe (which terms are defined herein below) and performing a two-step assay for detection of antibodies to Trypanosoma cruzi, HTLV-1, HTLV-2, HIV-1 and HIV-2, a greater or comparable readout signal is generated, as compared to a three-step assay. With respect to a greater signal, such a signal enhances assay performance which improves assay sensitivity.
More specifically, T. cruzi is the causative agent of Chagas' disease, a major public health problem in Latin America and growing concern in the United States, as the number of infected immigrants increases. There is currently no testing of U.S. blood products for T. cruzi infection. The best tests available, although highly sensitive, are not of high enough specificity to be useful for widespread screening of the blood supply in this country. Thus, the present invention provides a much needed immunoassay for detection of T. cruzi antibodies.
Human Immunodeficiency Virus Type I and Type II (HIV-1 and HIV-2, respectively) are the cause of a debilitating and lethal disease referred to as Autoimmune Deficiency Syndrome (AIDS). Since the viruses may be carried in blood or plasma, assays are required which are able to detect infected, donated blood or plasma in order to prevent recipients from contracting the disease. Further, assays are also required for the detection of HIV-1 and HIV-2 in infected individuals. The present assay allows for the detection of antibodies to these two deadly viruses.
Human T-Lymphotropic Virus Type I and Type II (HTLV-1 and HTLV-2, respectively) are retroviruses that appear to play a role in human cancers. These viruses may also be carried in the blood or plasma; thus, it is important that blood and plasma donations be screened in order to prevent transmission to susceptible donors. It is also important that those individuals infected with these viruses be diagnosed properly. The present assay allows for the detection of antibodies to HTLV-1 and HTLV-2.
All U.S. patents and publications referred to herein are hereby incorporated in their entirety by reference.